RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner. The RNAi machinery can be harnessed to destruct any mRNA of a known sequence. This allows for suppression (knock-down) of any gene from which it was generated and consequently preventing the synthesis of the target protein. Smaller siRNA duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Bartel, D. P. Cell 2000, 101, 25-33). Synthetic RNA duplexes can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control.
Chemical modification of RNA duplexes leads to improved physical and biological properties such as nuclease stability (Damha et al., Drug Discovery Today, 2008, 13(19/20), 842-855), reduced immune stimulation (Sioud TRENDS in Molecular Medicine, 2006, 12(4), 167-176), enhanced binding (Koller, E. et al., Nucl. Acids Res., 2006, 34, 4467-4476), enhanced lipophilic character to improve cellular uptake and delivery to the cytoplasm.
Since robust chemistry is a prerequisite for biological studies, development of efficient and reproducible methods for preparation of various oligonucleotide conjugates is of considerable importance (Harri Lönnberg, Bioconjugate Chemistry, 2009, 20, 1065-1094).
Chemically modified siRNA may be used as therapeutics to improve siRNA efficacy. In principle, chemically modified siRNA may be used to overcome efficacy related problems such as half-life in vivo, biodistribution and potency (Gaynor, J. W.; Campbell, B. J.; Cosstick, R. Chem. Soc. Rev., 2010, 39, 4169-4184).
Chemical modifications of RNA have relied heavily on work-intensive, cumbersome, multi-step syntheses of structurally novel nucleoside analogues and their corresponding phosphoramidites prior to RNA assembly. In particular, a major emphasis has been placed on chemical modification of the 2′-position of nucleosides. A rigorous approach to structure-activity-relationship (SAR) studies of chemical modifications will obviously require synthesis and evaluation of all four canonical ribonucleosides [adenosine (A), cytidine (C), uridine (U), guanosine (G)]. Furthermore, some chemical modifications bear sensitive functional groups that may be incompatible with state-of-the-art automated synthesis of RNA as well as subsequent downstream cleavage-deprotection steps. These attributes have made chemical modification of RNA prior to synthesis rather low-throughput and limited in scope.
Post-synthetic chemical modifications of RNA have centered for the most part on simple conjugation chemistry. Conjugation has largely been performed on either the 3′- or the 5′-end of the RNA via alkylamine and disulfide linkers. These modifications have allowed conjugation of RNA to various compounds such as cholesterol, fatty acids, poly(ethylene)glycols, various delivery vehicles and targeting agents such as poly(amines), peptides, peptidomimetics, and carbohydrates.
As 2′-OH is not required for siRNA to enter the RNAi pathway (Chiu, Y-L.; Rana, J. M. RNA, 2003, 9, 1034-1048), the 2′-position of ribose ring in siRNA is a common target for chemical modifications.
Methods for forming azido-modified nucleic acid conjugates of reporter molecules, carrier molecules or solid support utilizing “click” chemistry are disclosed in US 2008/0050731.
Synthesis of modified RNA and DNA utilizing an alkyne handle on a base and subsequent “click chemistry” is disclosed in WO 2008/052775 and in CN 101550175. Chemical modification of siRNA at the 2′-position using “click” chemistry is disclosed in WO 2011/0990968.
Recent reviews regarding “click” chemistry and oligonucleotide synthesis are covered by Gramlich et al., Angew. Chem. Int. Ed., 2008, 47, 8350-8358; Amblard et al., Chem. Rev., 2009, 109, 4207-4220.
Sequential bis-conjugation of oligonucleotides using click-oxime and click-Husigen protocols was reported by Defrancq et al. JOC, 2010, 75, 3927-3930.
There remains a need for a post synthetic method for modifying RNA molecules that can provide one or more of the following benefits: 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cleavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format; and 6) allows for an efficient orthogonal post-synthetic chemical modifications at multiple sites